Cloning, sequencing, and characterization of the bifunctional xylosidase-arabinosidase from the anaerobic thermophile Thermoanaerobacter ethanolicus

The gene for the bifunctional xylosidase-arabinosidase (xarB) from the ther mophilic anaerobe Thermoanaerobacter ethanolicus JW200 was cloned, sequence d, and expressed in Escherichia coli (Genebank Accession No. AF135015). Ana lysis of the recombinant enzyme revealed activity against multiple substrat es with the highest affinity towards p-nitrophenyl beta-D-xylopyranoside (p NPX) and highest activity against p-nitrophenyl alpha-L-arabinopyranoside ( pNPAP), respectively. Thus, we classify this enzyme as a bifunctional xylos idase-arabinosidase. Even though both sequences are 96% identical on the am ino acid level, excluding the amino-terminal end, a frame-shift mutation in the 5' region of the gene in T. brockii ATCC 33075 and a deletion in a dow nstream open reading frame in T, ethanolicus seem to have occurred through evolutionary divergence of these two species. This represents an interestin g phenomenon of molecular evolution of bacterial species, as PCR analysis o f the region around the deletion indicates that the deletion is not present in T. brockii sp. finnii and T. brockii sap. brockii type strain HTD4. (C) 2000 Elsevier Science B.V. All rights reserved.